Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bone morphogenetic protein 7 |
| Abbreviated Name | BMP-7 |
| SCOP Family | Amyloid peptides |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P18075 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 140 |
| Molecular Weight | 15680.7 |
| Pi | 8.47 |
| Molecular Weight | 15680.7 |
| Disulphides | Unknown |
| Full Sequence |
STGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lee DH, Baek HS,Lee MH, Park JC (2005) Current Applied Physics, 5, 422-425 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | n/a |
| Expression Protocol | After transformation of E. coli BL21 (DE3) (Novagen, WI, USA), BMP-7 synthesis was induced by addition of 1 mM isopropyl-thio-b-D-glucopyranoside. The BMP-7 was prepared as inclusion bodies produced in E. coli BL21 (DE3). Inclusion bodies were harvested and washed several times with PBS and dH2O. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | PBS and dH2O |
| Solubilization Buffer | 5% (w/v) solution of n-cethyltrimethylammonium chloride (CTAC) in 0.1 M Tris–HCl, pH 9.1, containing 0.25 M b-mercaptoethanol (b-ME), 75 lg/ml PMSF, 7 |
| Refolding Buffer | 1 mM EDTA, 3.5 M urea, 10–100 mM b-ME in 20 mM Tris–HCl, pH 9.1 |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 9.1 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 10-100mM |
| Refolding Protocol | Inclusion bodies were harvested and washed several times with PBS and dH2O. Inclusion bodies were solubilized using a 5% (w/v) solution of n-cethyltrimethylammonium chloride (CTAC) in 0.1 M Tris–HCl, pH 9.1, containing 0.25 M B-mercaptoethanol (b-ME), 75 lg/ml PMSF, 75 lg/ml leupeptin, and 75 lg/ml benzamidine at 50 °C for 1 h. The reducing agent was then removed by size exclusion chromatography using G-25 Sephadex gel filtration columns (PD-10, desalting columns; Pharmacia-LKB, USA) and refolding initiated by dilution into refolding buffer (1 mM EDTA, 3.5 M urea, 10–100 mM b-ME in 20 mM Tris–HCl, pH 9.1). Surfactant free rhBMP-7 (0.5 mg/ml) was subsequently renatured by dialyzing against the refolding buffer at 4 °C overnight, with gentle agitation to promote oxidation. The dialyzed solution was concentrated with ultrafiltration kit (Millipore, Corp., MA, USA) and purified by gel filtration chromatography (Shodex OHpak SB-803 column; Showa Denko K.K., Tokyo, Japan). |
| Refolding Assay | Biological assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |